As to multi-layered, chemical analysis materials (sheet form) enabling analysis through dry processes, those which comprise a support having provided thereon in sequence a single- or double-layered reagent layer and a non-fibrous, isotropic, sample-spreading layer are known as described, for example, in Japanese Patent Publication No. 33800/74 (corresponding to U.S. Pat. No. 3,630,957), Japanese Patent Application (OPI) Nos. 53888/74 (corresponding to U.S. Pat. No. 3,992,158), 137192/75 (corresponding to U.S. Pat. No. 3,977,568), 40191/76 (corresponding to U.S. Pat. No. 4,042,335), 3488/77 (corresponding to U.S. Pat. No. 4,066,403), 89796/78 (corresponding to U.S. Pat. No. 4,069,017), and 131089/78 (corresponding to U.S. Pat. No. 4,144,306). When an aqueous liquid sample is applied to the sample-spreading layer of these materials, the aqueous liquid sample migrates into the reagent layer with a uniform concentration, and causes a color-forming reaction to occur there. The changes in color density which occurs enables the concentration or level of a certain component in the aqueous liquid sample to be determined.
In determining a certain component in an aqueous liquid, a single reagent very often can not be employed for the chemical analysis and, in may cases, several reagents are used in combination. For example, in determining urea in blood, a direct determination of urea with a single reagent ordinarily is not used. Generally, urea is determined through several step reactions; for example, urea is first enzymatically decomposed with a first reagent, urease, to produce ammonia, and the ammonia is in turn brought into contact with a second reagent, for example, a pH indicator or a mixture of a diazonium salt and a coupler, thus eventually the presence of urea being determined through the dye formation system.
Multi-layered, analysis sheets using a plurality of reagents for multi-step reactions as described above include those wherein all necessary reagents can be incorporated in a single reagent layer, and those where the reagents are conveniently separated into a primary reagent and a secondary reagent depending on the order of the reactions, each of which reagents is incorported in, or forms, a different reagent layer.
Furthermore, multi-layered, chemical analysis sheets described in U.S. Pat. No. 3,011,874 and Japanese Patent Application (OPI) No. 3488/77 have a complicated multi-layered structure wherein reagents are incorporated separately into two layers, with one reagent layer using a hydrophobic binder or covered with a hydrophobic membrane for protection against permeation of water. Thus, reactions for determining an component in an aqueous liquid sample are clearly separated into two stages--first, aqueous reaction; then, non-aqueous reaction.
Multi-layered, chemical analysis sheets having the above-described multi-layered construction are generally prepared by repeating the steps of coating of a hydrophilic binder layer on a hydrophobic binder layer. This often results in delamination due to weak adhesion between the two layers. Therefore, in practice, at least one adhesive layer, called an subbing layer or an interlayer, is provided on the hydrophobic binder layer, followed by providing thereon the hydrophilic binder layer. Thus, to provide a reagent layer as two layers, a hydrophobic layer and a hydrophilic layer, requires production steps which are quite complicated.
In an analysis procedure for determining a specific component in an aqueous solution sample, which requires a plurality of reagents separated into two or more groups and one group of reagents to react in a hydrophilic environment and the other group of reagents in a non-aqueous environment, conventionally well known techniques utilize multi-layered, chemical analysis materials having a complicated construction wherein the reagents are incorporated separately in a hydrophilic binder layer and a hydrophobic binder layer. On the other hand, in the multi-layered chemical analysis material of the present invention for analyzing an aqueous liquid sample the reagent layer of the analysis material has a single- or multi-layered structure supported by a hydrophilic binder. Thus the coating and, therefore, production steps are simplified.